This disclosure relates to methods of preparation and processing of biological samples. More particularly, the disclosure relates to the processing of biological samples to be used for performing diagnostic assays and for therapeutic uses.
Despite unprecedented progress in measurement techniques over the recent years, satisfactory noninvasive measurements of target analytes in biological samples are still not possible in most cases. Generally, one or more sample pretreatment steps are necessary. These steps are referred to as sample preparation, the goal of which is to render a raw sample suitable for a measurement with a satisfactory signal to noise ratio. The sample preparation is accomplished by proceeding through cleanup, enrichment or concentration, and medium balance steps. In addition, in the case of measuring cellular contents, the molecules of interest must be released to the medium via cell lysis. Frequently, a lysate treatment step, involving various reagents, is needed to make the lysate assay-ready by modifying the target and non-target molecules or adjusting the lysate composition. Sample preparation is often the bottleneck in the measurement process, as it tends to be slow and generally involves multiple reagents and manual steps that require substantial time, complexity and cost.
The complexity of sample preparation can be better appreciated by referring to a typical example in which pathogenic bacteria in human urine are identified through rRNA hybridization where a specific sequence on the 16S rRNA is hybridized with a labeled complementary nucleic acid probe. An exemplary sample preparation protocol employs the foretold five steps as follows: 1) relatively large particles, such as crystals, and excess ions are removed (cleanup); 2) the bacteria count per unit volume is increased by reducing the water (liquid) content (enrichment); 3) the ribosomes are released by lysing the cells (lysis); 4) the lysate is treated such that the rRNA is partially untangled from the accompanying proteins and its conformation is modified to better expose the target region to the probes (lysate treatment); and 5) the chemical and ionic composition of the lysate is adjusted to support hybridization (medium balance).
In some instances, the sample preparation is further complicated by the need to process the sample with various reagents. For example, reagents may be needed for lysis, binding and elution, precipitation, removal or inhibition of interferants and/or contaminants, denaturing of DNA to obtain single stranded DNA, separation of rRNA from ribosomal proteins, and denaturing of enzymes or other proteins such as DNAse and RNAse. One example of a reagent treatment for the purification of nucleic acids involves cell lysis and molecular denaturation by a chaotropic agent (guanidinium thiocyanate) followed by nucleic acid extraction by phenol-chloroform liquid-liquid separation, which is a complex method involving very hazardous reagents. Further, sample processing by reagents may involve additional steps such as heating and bead milling, for example, to enhance the function and effectiveness of reagents.